Simplified nuclei analysis platform and biomarker matrix that supports genotoxic mode of action determinations

ABSTRACT

The present invention relates a simple method for evaluating free eukaryotic cell nuclei for biomarkers of DNA damage and/or transcription factor activation, activity, or expression levels and/or epigenetic modifications to chromatin or chromatin-associated factors. The invention also teaches useful strategies for combining nuclear biomarkers into a matrix of endpoints that are capable of elucidating genotoxicants&#39; primary mode of DNA-damaging activity. Kits for conducting methods according to the invention are also described.

This application is a continuation of U.S. Patent Application Serial No.14/201,138, filed Mar. 7, 2014, which claims the priority benefit ofU.S. Provisional Patent Application Ser. No. 61/775,494, filed Mar. 9,2013, which are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to a simple method for evaluating free eukaryoticcell nuclei for biomarkers of DNA damage and/or transcription factoractivation, activity, or expression levels and/or epigeneticmodifications to chromatin or chromatin-associated structures. Theinvention also teaches useful strategies for combining nuclearbiomarkers into a matrix that is capable of determining genotoxicants'primary mode of DNA-damaging activity.

BACKGROUND OF THE INVENTION

The cell nucleus is a membrane-bound organelle found in eukaryoticcells. The nucleus houses the majority of each cell's nucleic acidcontent, and is therefore considered the control center that directsgene expression and protein synthesis. The long, linear, double-helixDNA molecules are ordinarily maintained in structures calledchromosomes, and histones as well as other chromatin-associatedstructures are known to play an important role in maintaining thishigher order configuration.

Nuclei and the processes that they govern are of extreme importance tobiological sciences. Methods for analyzing nuclei and the function ofthe nucleic acids they contain are extremely diverse, and include stabletransfection, gene knock-out and knock-in techniques, western blotting,site-directed mutagenesis, DNA sequencing, electron microscopy, imageanalysis, and polymerase chain reaction (PCR). The list continues togrow given the importance of cells' genetic material.

Indeed, many lines of investigation benefit from an understanding of howcells and nuclei in particular react to certain stimuli. For instance,biomarkers of DNA damage can be useful for determining whether a testchemical is DNA-reactive and therefore likely to be mutagenic. Nucleiare especially well suited to make these assessments since they housethe bulk of cells' genetic material, and numerous DNA damage-responsivepathways within this organelle have been described.

Responses to DNA damage typically involve soluble protein factors thatreside within the cytosol and/or the nucleus. Upon DNA damage, some areactivated via phosphorylation, some become translocated from cytosol tonucleus, and others are controlled in other manners, for instancecleavage which activates an enzymatic function. These types of responsescan be studied with antibodies or other high affinity reagents thatspecifically recognize altered DNA and/or proteins that have beentranslocated to the nucleus, activated, or otherwise modified to dealwith the damage. The literature is full of techniques that are capableof studying these types of activities, and include western blotting,cleavage of luminescent substrates, electrophoretic mobility shiftassays, image analysis, and flow cytometry, among others. Image analysisand other visual assessments based on microscopy tend to call for cellfixation, antibody labeling, and washing steps. Similarly, flowcytometric approaches tend to specify several processing steps wherebyantibodies specific for DNA damage or proteins associated with damageare applied to fixed cells or liberated nuclei, followed by removal ofunbound fluorescent reagents via a centrifugation or similar steps. Formany types of analyses, heat and/or other strong denaturing conditionsare applied to provide the antibody(s) with greater access tonuclei-associated epitopes. For many laboratory environments, especiallywhere higher throughput is required, it would be preferable to utilize aso-called “homogeneous assay” whereby cells are simply brought intocontact with one solution and then analyzed without the need for furthersample processing steps.

The present invention overcomes the disadvantages of prior artapproaches, and satisfies the need of establishing robust, reliable,high throughput methods for evaluating nuclei for biomarkers of DNAdamage and/or transcription factor activation, activity, or expressionlevels and/or epigenetic modifications to chromatin orchromatin-associated factors.

SUMMARY OF THE INVENTION

As used herein, the terms “nuclei”, “nuclei events”,“detergent-liberated nuclei”, and “free nuclei” are used interchangeablyto describe chromatin and other nuclear factors that are surrounded by anuclear membrane and that have been liberated from cells through contactwith one or more cell lysis (e.g., detergent-containing) solutions.

As used herein, these terms, “nuclei”, etc., are also inclusive ofbundles of metaphase chromosomes that lack a nuclear envelop butnone-the-less remain together in an aggregated state upon contact withthe one or more cell lysis solutions. Importantly, the aggregation isnot excessive whereby multiple cells' metaphase chromosomes are found toclump together, rather these bundles of metaphase chromosomes remain inunits that correspond to single cells' metaphase chromosomes. Withoutbeing bound by belief, it is believed that the most likely explanationfor this advantageous state of aggregation is that upon digestion ofcytoplasmic membranes, cytosolic and/or metaphase-associated fibersenvelop the metaphase chromosomes, resulting in maintenance of theirchromosome complement.

As used herein, the term “chromatin debris” is used to describechromatin that is associated with dead and/or dying cells. In theseinstances, the chromatin may or may not be bound by a nuclear envelope,and the chromatin may or may not exist in typical amounts. For instance,apoptotic bodies will generally have sub-2n DNA content owing to thefragmentation of nuclei that occurs during this process of cell death.None-the-less, there are some applications of the current invention thatwould benefit from analysis of these particles for nuclei-associatedepitopes, and in these cases the term “chromatin debris” is used.

As used herein, “NAESA/L” refers to fluorescent nuclei-associatedepitope-specific antibody or other high affinity ligand, includingantibody fragments that retain binding specificity to thenuclei-associated epitope of interest, as well as polypeptide antibodymimics or nucleic acid aptamers that exhibit binding specificity for thenuclei-associated epitope of interest.

A first aspect of the invention relates to a method for analyzingdetergent-liberated nuclei and/or chromatin debris for nuclei-associatedepitopes. The method includes: contacting a sample containing eukaryoticcells with a solution comprising one or more cell lysis reagents, afluorescent nucleic acid dye (NAD), and one or more NAESA/L, the NAD andone or more NAESA/L having distinct fluorescent emission spectra, saidcontacting being effective to digest eukaryotic cell cytoplasmicmembranes but not nuclear membranes, aggregate metaphase chromosomes ofa single cell into a single bundle of chromosomes, label chromatin withthe NAD, and label the one or more nuclei-associated epitopes with theone or more NAESA/L; exciting the NAD and one or more NAESA/L with lightof an appropriate excitation wavelength; detecting fluorescent emissionand light scatter produced by the nuclei and/or chromatin debris andcounting one or more of the following events: the number of nuclei, thenumber of nuclei positively labeled by the one or more NAESA/L, thenumber of chromatin debris, the number of chromatin debris positivelylabeled by the one or more NAESA/L, the number of nuclei in G1, S, andG2/M phases of the cell cycle, and the number of polyploid nuclei; anddetermining one or more of the following measurements: the frequency ofnuclei positively labeled by the one or more NAESA/L relative to totalnuclei, the frequency of chromatin debris positively labeled by the oneor more NAESA/L relative to total chromatin debris and/or total nuclei,the proportion of nuclei in G1, S, and G2/M phases of the cell cycle,the proportion of polyploid nuclei, and mean and/or median fluorescenceof the one or more NAESA/L.

A second aspect of the present invention relates to a method forassessing the response of nuclei-associated biomarkers of DNA damageand/or transcription factor activation, activity, or expression levelsand/or epigenetic modifications resulting from a chemical or physicalagent. This method includes: exposing eukaryotic cells, previouslyexposed to a chemical or physical agent, to a solution comprising one ormore cell lysis reagents, optionally RNase, a NAD, and one or moreNAESA/L, the NAD and one or more NAESA/L having distinct fluorescentemission spectra, said exposing being effective to digest eukaryoticcell cytoplasmic membranes but not nuclear membranes, aggregatemetaphase chromosomes of a single cell into a single bundle ofchromosomes, label chromatin with the NAD, and label the one or morenuclei-associated epitopes with the one or more NAESA/L; exciting theNAD and one or more NAESA/L with light of an appropriate excitationwavelength; detecting fluorescent emission and light scatter produced bythe nuclei and/or chromatin debris and counting one or more of thefollowing events: the number of nuclei, the number of NAESA/L-positivenuclei, the number of chromatin debris, the number of NAESA/L-positivechromatin debris, the number of nuclei in G1, S, and G2/M phases of thecell cycle, and the number of polyploid nuclei. Changes to theproportion of NAESA/L-positive nuclei relative to total nuclei and/orchanges to the proportion of NAESA/L-positive chromatin debris relativeto total chromatin debris and/or total nuclei indicates that thechemical or physical agent affected the nuclear biomarker. Anothervaluable endpoint that is relevant for some nuclei biomarkers is ameasure of central tendency of NAESA/L-associated fluorescence. In thecontext of assessing a chemical or physical agent, the change (i.e., the“shift”) in mean and/or median fluorescence of the one or more NAESA/Lfor an exposed sample, compared to unexposed or negative controleukaryotic cells, indicates that the chemical or physical agent affectedthe nuclear biomarker.

A third aspect of the present invention relates to a method of assessingthe toxicity of a chemical or physical agent. The method includes:exposing eukaryotic cells to a chemical or physical agent and performingthe method according to the first aspect of the invention wherein, incomparison to unexposed or negative control eukaryotic cells, asignificant change to the proportion of the one or more NAESA/L-positivenuclei relative to total nuclei and/or change to the proportion of theone or more NAESA/L-positive chromatin debris relative to totalchromatin debris and/or total nuclei indicates that the chemical orphysical agent affected the nuclei-associated epitope of interest; asignificant change to the proportion of nuclei in one or more phases ofthe cell cycle and/or in the proportion of polyploid nuclei indicatesthat the chemical or physical agent perturbed the cell cycle; and asignificant change in the one or more NAESA/L-positive nuclei and/orchromatin debris indicate that the chemical or physical agent affectedthe nuclei-associated epitope of interest, whereby an effect of thechemical or physical agent on two or more of these endpoints allows adetermination of the type of toxicity caused by the chemical or physicalagent.

In accordance with these aspects, and without limitation thereto, incertain embodiments the one or more endpoints may include one or more ofan increase in the phosphorylated histone 2AX (γH2AX), which indicatesclastogenic activity; an increase in the phosphorylated histone 3 (H3)or polyploidy nuclei, each of which indicates aneugenic activity; anincrease in cleaved poly(ADP-ribose) polymerase (PARP)+ events, cleavedcaspase 3 (Cas 3)+ events, cleaved caspase 7 (Cas 7)+ events, cleavedcaspase 9 (Cas 9)+ events, tetramethylrhodamine ethyl ester(TMRE)-events, ethidium monoazide bromide (EMA)+, or propidium monoazidebromide (PMA)+ events indicates cytotoxicity; and a reduction in nucleito bead ratios or nuclei to time ratios, or ATP levels indicatescytotoxicity.

A fourth aspect of the present invention is related to a panel ofNAESA/Ls that can be used singularly or in combination to achieveseveral measurements that are valuable for discriminating genotoxicagents' primary mode of action. In performing the method according tothe first, second, or third aspects of the invention using a singularNAESA/L, then additional NAESA/Ls can be used in parallel to assessother epitopes. Alternatively, multiple compatible NAESA/L can be usedin a single sample for simultaneous measurement of NAESA/L biomarkers.The NAD and NAESA/L are excited with light of appropriate excitationwavelength(s), and any one or more, preferably two or more, ofabove-identified events or changes in proportions are determined. Ashift in an exposed sample's NAESA/L-associated fluorescence compared tounexposed or negative control eukaryotic cells may also be used toindicate that the chemical or physical agent affected the nuclearbiomarker. By considering the effect of chemical or physical agenttreatment on the two or more of these endpoints, a determination ofgenotoxic MOA is made, and discriminated from cytotoxic activity.

A fifth aspect of the present invention relates to a kit that includesone or more eukaryotic cell membrane lysis solutions; a NAD; and one ormore NAESA/L that bind specifically to a nuclei-associated epitope,preferably two or more NAESA/L that bind specifically to two or moredistinct nuclei-associated epitopes. The kit may optionally contain oneor more additional components and reagents, including a third NAESA/Lthat binds specifically to a third epitope associated with cytotoxicity;a reagent that is responsive to cytotoxicity (e.g., a reagent thatlabels a marker of mitochondrial health, for instance a mitochondrialmembrane potential dye, or is a dye that is otherwise responsive tocytotoxicity); RNase; counting beads; and a computer-readable mediumthat contains instructions for statistical analyses for characterizingin vitro genotoxicity as the result of clastogenicity, aneugenicity, orcytotoxicity based on the behavior of a eukaryotic cell in response toexposure to a chemical or physical agent and following exposure to thekit components. The kit may further include instructions for use of thekit components. In certain embodiments, the one or more eukaryotic cellmembrane lysis solutions also contain(s) all other reagents suppliedwith the kit.

A sixth aspect of the invention relates to a kit that includes one ormore eukaryotic cell membrane lysis solutions; RNase, a NAD, acomputer-readable medium that contains instructions for statisticalanalyses for characterizing toxicity as the result of clastogenicity,aneugenicity, or cytotoxicity based on the behavior of a eukaryotic cellin response to in vitro exposure to a chemical or physical agent andfollowing exposure to the kit components with one or more NAESA/L thatbind specifically to one or more nuclei-associated epitopes, preferablytwo or more NAESA/L that bind specifically to two or more distinctnuclei-associated epitopes as described above, and optionally a thirdNAESA/L that binds specifically to a third epitope associated withcytotoxicity or a reagent that is responsive to cytotoxicity (e.g., areagent that labels a marker of mitochondrial health, for instance amitochondrial membrane-potential dye or luminescent ATP-specificreagent, or is a reagent that is otherwise responsive to cytotoxicity).The kit may further include instructions for use of the kit components.In certain embodiments, the one or more eukaryotic cell membrane lysissolutions also contain(s) all other reagents supplied with the kit.

A seventh aspect of the invention relates to a reagent for performingthe methods of the present invention. The reagent is a composition inthe form of an aqueous solution that includes: (i) an effective amountof one or more agents for causing eukaryotic cell membrane lysis; (ii)optionally an effective amount of an RNase; (iii) an effective amount ofa NAD; (iv) optionally a suitable concentration of counting beads; and(v) an effective amount of one or more, two or more, or three or moreNAESA/L against nuclei-associated epitopes. In certain embodiments, theaqueous solution includes components (i)-(v) as identified above. Incertain other embodiments, where the NAD is a DNA-specific dye, then theeffective amount of RNase, component (ii), can be omitted.

In the several aspects of the invention, particularly preferredcombinations of reagents have been identified that are capable of highlyselective discrimination of cytotoxicity, genotoxicity with clastogenicmode of action, and genotoxicity with aneugenic mode of action.Preferably, each of these combinations includes, without limitation, aNAD, a first NAESA/L that binds specifically a nuclei-associated epitopeassociated with double-strand DNA breaks; a second NAESA/L that isresponsive to aneugenic activity; and either a third NAESA/L that bindsspecifically to a third epitope associated with cytotoxicity or areagent that is responsive to cytotoxicity (e.g., a reagent that labelsa marker of mitochondrial health, for instance a mitochondrialmembrane-potential dye or luminescent ATP-specific reagent, or is areagent that is otherwise responsive to cytotoxicity). Particularlypreferred combinations of reagents include, without limitation, (i) aNAD, a first NAESA/L that binds specifically to an epitope present onphosphorylated γH2AX, a second NAESA/L that binds specifically to anepitope present on phosphorylated H3, and a third NAESA/L that bindsspecifically to an epitope present on cleaved caspase 3 or cleaved PARP;(ii) a NAD, a first NAESA/L that binds specifically to an epitopepresent on phosphorylated γH2AX, a second NAESA/L that bindsspecifically to an epitope present on phosphorylated H3, and a DNA dyethat penetrates dead and dying cells, but not viable cells, such as thephotoactivatable dyes EMA or PMA; (iii) a NAD, a first NAESA/L thatbinds specifically to an epitope present on phosphorylated γH2AX, asecond NAESA/L that binds specifically to an epitope present onphosphorylated H3, and a mitochondrial membrane dye such as TMRE; (iv) aNAD, a first NAESA/L that binds specifically to an epitope present onphosphorylated γH2AX, a second NAESA/L that binds specifically to anepitope present on phosphorylated H3, and a reagent that measurescellular ATP levels; (v) a NAD, a first NAESA/L that binds specificallyto an epitope present on cleaved PARP, and a second NAESA/L that bindsspecifically to an epitope present on cleaved caspase 3 and/or 7 and/or9; and (vi) a NAD, a first NAESA/L that binds specifically to an epitopepresent on phosphorylated-H3, and either a second NAESA/L that bindsspecifically to an epitope present on Ki-67 or proliferating cellnuclear antigen (PCNA) or the dye carboxyfluorescein N-succinimidylester (CFSE), which are markers of cell proliferation. In addition tothe foregoing combinations, counting beads can be incorporated into thecombinations to provide information on absolute nuclei (and, thus, cell)counts.

The methods described herein provide for the assessment of eukaryoticcell nuclei for biomarkers of DNA damage and/or transcription factoractivation, activity, or expression levels and/or epigeneticmodifications to chromatin or chromatin-associated structures using,preferably, flow cytometry, image analysis, imaging flow cytometry, orlaser-scanning cytometry. The invention also teaches useful strategiesfor combining nuclear and other biomarkers into a matrix that is capableof determining genotoxicants' primary mode of DNA-damaging activity. Theprimary advantage of this methodology relative to other procedures isthat these assessments are made very efficiently, through the use of aso-called homogenous (combine and read) assay. The need for such ahomogenous assay is met by providing a detergent-based solution thatcombines cytoplasmic membrane lysis, retention of nuclear envelopes,degradation of RNA, pan-chromatin fluorescent staining, and optionallythe fluorescent labeling of nuclei-associated epitope(s) of interest inone simple step. This minimizes time and effort spent handling a samplefor evaluation, and therefore affords significant savings in acquiringthe much-needed information for evaluating nuclei biomarkers for DNAdamage, transcription factor activity, activation, or expression levels,or epigenetic modifications to chromatin or chromatin-associate factors.Furthermore, the accompanying Examples demonstrate methods for buildinga matrix of nuclei biomarkers that effectively elucidate genotoxic modeof action by facilitating characterization of in vitro cytogeneticdamage resulting from clastogenic activity, aneugenic activity, or aconsequence of cytotoxicity. Importantly, the methods and kits describedherein provide for simultaneous analysis of multiple endpoints, that is,in a “multiplexed” manner, a characteristic that greatly enhances assayefficiency and information content. This, too, is demonstrated in theaccompanying Examples where the multiplexed analysis of severalendpoints affords comprehensive and efficient acquisition of severalnuclei-associated endpoints in a single assay format.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows flow cytometric bivariate plots of TK6 cells treated withsolvent (DMSO), the apoptosis-inducing agent anti-FAS, or the clastogenmethyl methanesulfonate (MMS). The X-axis corresponds to fluorescenceassociated with the nucleic acid dye SYTOX® Green, and the Y-axis showsfluorescence associated with anti-γH2AX. Following MMS treatment,nuclei's median anti-γH2AX-associated fluorescence intensity is visiblyincreased. This can be expressed as change in mean or mediananti-γH2AX-associated fluorescence intensity relative to solventcontrol. The apoptosis-inducing agent anti-FAS does not have this effecton TK6 cells. Note that in the case of the γH2AX biomarker, it ispreferable to only consider fluorescence for those events that fallwithin rectangular regions, as shown here. Higher anti-γH2AX-associatedfluorescence intensity, i.e., beyond the rectangles, tends to be due toapoptotic cells as opposed to cells with clastogen-induced double-strandDNA breaks. Also note that the dashed lines have been added forillustrative (reference) purposes only.

FIG. 2 shows flow cytometric bivariate plots of TK6 cells treated withsolvent (DMSO) or the apoptosis-inducing agent carbonyl cyanidem-chlorophenyl hydrazone (CCCP). The X-axis corresponds to fluorescenceassociated with the nucleic acid dye SYTOX Green, and the Y-axis showsfluorescence associated with anti-cleaved caspase 3. Following CCCPtreatment, the percentage of nuclei exhibiting anti-cleaved caspase3-positive events is visibly increased.

FIG. 3 shows flow cytometric bivariate plots of TK6 cells treated withsolvent (DMSO), the aneugen vinblastine, or the clastogen MMS. TheX-axis corresponds to fluorescence associated with the nucleic acid dyeSYTOX® Green, and the Y-axis shows fluorescence associated withanti-phospho-H3. Following vinblastine treatment, the percentage ofnuclei exhibiting anti-phospho-H3-positive events is increased relativeto solvent control. The clastogen MMS does not have this effect on TK6cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method for evaluating freeeukaryotic cell nuclei for biomarkers of DNA damage and/or transcriptionfactor activation, activity, or expression levels and/or epigeneticmodifications to chromatin or chromatin-associated factors. Theinvention also teaches useful strategies for combining nuclear and otherbiomarkers into a matrix that is capable of determining genotoxicants'primary mode of DNA-damaging activity.

One aspect of the present invention relates to a method for analyzingdetergent-liberated nuclei and/or chromatin debris for nuclei-associatedepitopes. The method involves contacting a sample containing eukaryoticcells with a detergent-containing solution that digests outercytoplasmic membranes, but not nuclear membranes, and that aggregatesmetaphase chromosomes into bundles of chromosomes. Thedetergent-containing solution contains RNase to effectively degrade RNA,a fluorescent nucleic acid dye (NAD) to label all chromatin, and one ormore nuclei-associated epitope-specific antibodies or other highaffinity ligands (NAESA/L) that specifically recognize nuclei-associatedepitope(s) of interest. The NAD and NAESA/L fluorescent reagents areexcited with light of appropriate excitation wavelength(s). Thefluorescent emission and light scatter produced by the nuclei and/orchromatin debris are detected. Any one or more of the following eventscan be counted: the number of nuclei, the number of NAESA/L-positivenuclei, the number of chromatin debris, the number of NAESA/L-positivechromatin debris, the number of nuclei in G1, S, and G2/M phases of thecell cycle, the number of polyploid nuclei, and NAESA/L-positive nucleifluorescence intensity.

A number of different endpoints can be determined from these counts,including: the frequency of NAESA/L-positive nuclei relative to totalnuclei, and the frequency of NAESA/L-positive chromatin debris relativeto total chromatin debris and/or total nuclei, the proportion of nucleiin G1, S, and G2/M phases of the cell cycle, the proportion of polyploidnuclei, and mean and/or median NAESA/L-associated fluorescenceintensity.

As indicated above, the frequency of NAESA/L-positive nuclei, chromatindebris, polyploid nuclei, etc., can be expressed relative to otherpopulations, for instance NAESA/L-positive nuclei can be expressedrelative to total nuclei. Alternatively, these populations can beexpressed per unit volume of sample or per unit time (based on thefluidic rate and the time taken to analyze the sample).

Alternatively, counting beads can be added to the sample and thefluorescent emission and light scatter of the counting beads is detectedand enumerated along with the other events to obtain an event-to-beadratio. When utilized, the counting beads can be included in the one ormore lysis solution(s) or separately introduced to the sample before orafter introduction of the one or more lysis solution(s). The countingbeads can be a suspension of relatively uniform particle (e.g., formedof latex or a polymer) that can be readily differentiated from thecells. Preferred counting beads include, without limitation,COUNTBRIGHT™ Absolute Counting Beads and 6 micron PEAK FLOW™ fluorescentmicrospheres from Life Technologies, and SPHERO™ multi-fluorophore beadsfrom Spherotech Inc. In one embodiment of the present invention, suchcounting beads are added along with the NAD and NAESA/L-containing lysissolution. However, it will be appreciated by those knowledgeable in theart that there are alternate and equally acceptable times during theprocedure when counting beads can be added and used effectively toobtain the desired values.

In certain embodiments, an additional reagent can be introduced that isresponsive to cytotoxicity. In one embodiment, this additional reagentis in the form of a DNA dye that penetrates dead and dying cells, butnot viable cells, such as the photoactivatable dyes ethidium monoazidebromide or propidium monoazide bromide (see U.S. Pat. Nos. 7,445,910 and7,645,593 to Dertinger et al., which are hereby incorporated byreference in its entirety), a mitochondrial membrane potential dye, or areagent that measures cellular ATP levels, e.g., a luminescent reagentsuch as luciferase/luciferin (Promega's CELLTITER-GLO® kit). Exemplarymitochondrial membrane potential dyes include, without limitation,tetramethylrhodamine ethyl ester (TMRE); tetramethylrhodamine methylester (TMRM); 3,3′ dihexyloxacarbocyanine iodide (DiOC₆);5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanineiodide (JC-1); 3,3-dimethyl-α-naphthoxacarbocyanine iodide (JC-9);1,1′,3,3,3′,3′-hexamethylindodicarbocyanine iodide (DilC₁);nonylacridine orange; safranine O; or rhodamine-123.

Exemplary endpoints for assessing cytotoxicity include, withoutlimitation, NAD-positive nuclei to time ratios, which are responsive totreatment-related changes to cell densities when compared to negativecontrol cells; increased NAESA/L-fluorescence specific for cleaved PARPor any one of cleaved caspases 3, 7, and 9, which indicate activation ofcell apoptotic pathways; ethidium monoazide bromide (EMA)- or propidiummonoazide bromide (PMA)-positive events as an indicator of (low)membrane integrity; tetramethylrhodamine ethyl ester (TMRE) negativecells as an assessment of mitochondrial membrane potential, and totalATP as measured using luciferase/luciferin as an indicator of overallcell energy stores and reflect overall cell health.

Eukaryotic cells suitable for carrying out the methods of the presentinvention include any types of animal cells, preferably mammalian cells,as well as plant protoplasts. Exemplary animal cells suitable forcarrying out the methods of the present invention include, withoutlimitation, immortalized cell lines, as well as cells which have onlyrecently been harvested from animal species (e.g., primary cellcultures). The eukaryotic cells can be cultured in vitro.

Preferred primary cell cultures are those that divide in culture (i.e.,with appropriate growth media, which for some cell types requires theinclusion of cytokines and/or other factors such as mitogens). Exemplarycell types that can be screened easily using the methods of the presentinvention include, without limitation, blood-, spleen-, lymph node-, orthymus-derived lymphocytes, bone marrow-derived cells including stemcells, and hepatocytes.

Exemplary immortalized cell lines include, without limitation, TK6,AHH-1, WIL-2NS, HepG2, HepaRG™, HeLa, MCF-7, MCL-5, NIH-3T3, Jurkat,HL-60, A549, Raji, CHO-K1, V79, Vero, Hepalclc7, and L5178Y cells, aswell as induced pluripotent stems cells.

The NAD reagent can be any dye that permeates the nuclear envelope andimparts fluorescence to chromatin. Any suitable NAD with appropriateexcitation and emission spectra can be employed, such as propidiumiodine, ethidium bromide, 7AAD, DRAQ 5, DRAQ 7, DAPI, Hoechst 33258,Hoechst 33342, YO-PRO®-1, SYTOX® Green, SYBR® Green I, SYTOX® Red, SYTO®11, SYTO®12, SYTO®13, SYTO®59, BOBO®, YOYO®, and TOTO®. The need forcontact with RNase is eliminated when DNA-specific nucleic acid dyes areused, for instance as is the case for DAPI, Hoechst 33258, and Hoechst33342. Effective amounts of these dyes will vary depending on thefluorescent properties of the dye, but generally these dyes can beintroduced in an amount of about 0.1 μg/m1 to about 15 μg/ml.

The one or more lysis solutions can be any suitable lysis solution, orcombination thereof, for cell membrane lysis. Non-ionic detergents areparticularly desirable for use in the one or more lysis solutions.According to one embodiment, the lysis solution consists of NaCl,Na-Citrate, and octylphenyl-polyethylene glycol (IGEPAL®, Sigma) indeionized water. Alternative embodiments include, without limitation,0.1% to 10% of one or more of IGEPAL®, TritonX, Tween20, Tween80, andsaponin in buffered solution, e.g., PBS.

Suitable NAESA/L reagents specifically bind to nuclei-associatedepitopes and have a fluorescent emission spectrum that does notsignificantly overlap with the emission spectrum of the NAD. PreferredNAESA/L reagents are those that are responsive to DNA damage and/ortranscription factor activation, activity, or expression levels and/orepigenetic modifications to chromatin.

In accordance with these aspects of the present invention, exemplarycategories of NAESA/L target structures include, without limitation,histone and histone-like proteins as well as histone modifications,whether defined as post-translational or otherwise, markers of cells inmetaphase, markers of apoptosis, markers of DNA damagecheckpoints/response, transcription factors, DNA adducts, DNAmethylation sites, proteins associated with DNA methylation, proteinsassociated with histone modification, and markers of cell proliferation.

Exemplary histone and histone-like proteins include, without limitation,histone 1 (H1), histone 2A (H2A), histone 2A.Z (H2A.Z), histone 2AX(H2AX), histone 3 (H3), histone 4 (H4), centromere protein A (CENP-A),centromere protein B (CENP-B), centromere identifier (CID),heterochromatin protein (HP1). Exemplary histone modifications include,without limitation, acetylation, methylation, phosphorylation,ubiquitination, glycosylation, ADP-ribosylation, carbonylation, andSUMOylation. A number of commercially available NAESA/L are availableagainst these targets. Specific targets in this category and theirNAESA/L include, without limitation, phosphorylated γH2AX andanti-pSer139-γH2AX; phosphorylated-H3 and anti-pSer1O-H3,anti-pSer28-H3, or anti-pThrl l-H3; HpTGEKP motif and its antibodies;acetylated-H3 and anti-K27ac-H3; methylated-H3 and anti-K4me3-H3;ubiquinated H2B and anti-K12ub1-H2B.

An exemplary marker of cells in metaphase includes, without limitation,Mitotic Protein. A number of commercially available NAESA/L areavailable against these targets. One specific target and its NAESA/Lincludes, without limitation phosphorylated Mitotic Protein and thephospho-Ser/Thr-Pro Mitotic protein monoclonal #2 (MPM2).

Exemplary markers of apoptosis include, without limitation, cleavedPARP, and cleaved caspase 3, 7, or 9. A number of commercially availableNAESA/L are available against these targets.

Exemplary markers of DNA damage checkpoints/response include, withoutlimitation, ChK1 and ChK2, ATM, ATR, BRCA1, BRCA2, RAD51, and p53. Anumber of commercially available NAESA/L are available against thesetargets.

Exemplary transcription factor targets include, without limitation, cAMPresponse element-binding protein (CREB), CREB-binding protein (CBP),NFκB, aryl hydrocarbon receptor (AhR), and aryl hydrocarbon receptornuclear translocator (ARNT). A number of commercially available NAESA/Lare available against these targets.

Exemplary DNA adducts include, without limitation, 0-6-methylguanine,7-methylguanine, 8-oxo-deoxyguanosine, 1,N(2)-propane deoxyguanosines,and 8-oxo-7,8-dihydro-2′-deoxyguanosine. A number of commerciallyavailable NAESA/L are available against these targets.

Exemplary DNA methylation sites include, without limitation, 5-methylcytidine, 5-carboxylcytosine, 5-formylcytosine, 5-hydroxymethylcytosine,and 3-methylcytosine. A number of commercially available NAESA/L areavailable against these targets.

Exemplary proteins associated with DNA methylation include, withoutlimitation, DNA methyltransferase 1 (DNMT1), DNA methyltransferase 2(DNMT2), DNA methyltransferase 3A (DNMT3A), DNA methyltransferase 3B(DNMT3B), and DNA methyltransferase 3-like protein (DNMT3L), and methylCPG-binding proteins. A number of commercially available NAESA/L areavailable against these targets.

Exemplary proteins associated with histone modification include, withoutlimitation, histone acetyltransferase (HAT), histone deacetylase (HDAC),histone demethylase (HDME), and sirtuin 2. A number of commerciallyavailable NAESA/L are available against these targets. One specifictarget and its NAESA/L includes, without limitation, phosphorylatedSIRT2 and the anti-phospho-Ser331-SIRT2.

Exemplary markers of cell proliferation include, without limitation,proliferating cell nuclear antigen (PCNA) and Ki-67. A number ofcommercially available NAESA/L are available against these targets.Alternatively, the dye carboxyfluorescein succinimidyl ester (CFSE) canbe used as a marker of cell proliferation, because its fluorescence iscut roughly in half as cells divide.

NAESA/L in the form of antibodies that bind specifically to one or moreof the exemplary biomarkers that contain the nuclei-associated epitopesare commercially available. Also encompassed by the definition ofNAESA/L are binding portions of such antibodies, including monovalentFab fragments, Fv fragments (e.g., single-chain antibody, scFv), andsingle variable V_(H) and V_(L) domains, and the bivalent F(ab')₂fragments, Bis-scFv, diabodies, triabodies, minibodies, etc. Theseantibody fragments can be made by conventional procedures, such asproteolytic fragmentation procedures, as described in James Goding,MONOCLONAL ANTIBODIES:PRINCIPLES AND PRACTICE 98-118 (Academic Press,1983) and Ed

Harlow and David Lane, ANTIBODIES: A LABORATORY MANUAL (Cold SpringHarbor Laboratory, 1988); Houston et al., “Protein Engineering ofAntibody Binding Sites: Recovery of Specific Activity in an Anti-DigoxinSingle-Chain Fv Analogue Produced in Escherichia coli,” Proc. Natl.Acad. Sci. USA 85:5879-5883 (1988); Bird et al, “Single-ChainAntigen-Binding Proteins,” Science 242:423-426 (1988), which are herebyincorporated by reference in their entirety, or other methods known inthe art.

Antibody mimics can also serve as NAESA/L in the present invention. Anumber of antibody mimics are known in the art including, withoutlimitation, those known as monobodies, which are derived from the tenthhuman fibronectin type

III domain (¹⁰Fn3) (Koide et al., “The Fibronectin Type III Domain as aScaffold for Novel Binding Proteins,” J. Mol. Biol. 284:1141-1151(1998); Koide et al., “Probing Protein Conformational Changes in LivingCells by Using Designer Binding Proteins: Application to the EstrogenReceptor,” Proc. Natl. Acad. Sci. USA 99:1253-1258 (2002), each of whichis hereby incorporated by reference in its entirety); and those known asaffibodies, which are derived from the stable alpha-helical bacterialreceptor domain Z of staphylococcal protein A (Nord et al., “BindingProteins Selected from Combinatorial Libraries of an alpha-helicalBacterial Receptor Domain,” Nature Biotechnol. 15(8):772-777 (1997),which is hereby incorporated by reference in its entirety). Thesemonobodies and affibodies can be screened and selected for their bindingaffinity to one or more of the above-identified biomarkers that containthe nuclei-associated epitopes.

Another form of antibody mimic is a nucleic acid aptamer, which can beformed of DNA or RNA or modified nucleotides, and is characterized byspecificity for one or more of the above-identified biomarkers thatcontain the nuclei-associated epitopes. Aptamers are single-stranded,partially single-stranded, partially double-stranded, or double-strandednucleotide sequences, advantageously a replicatable nucleotide sequence,capable of specifically recognizing a selected non-oligonucleotidemolecule or group of molecules by a mechanism other than Watson-Crickbase pairing or triplex formation. Aptamers include, without limitation,defined sequence segments and sequences comprising nucleotides,ribonucleotides, deoxyribonucleotides, nucleotide analogs, modifiednucleotides and nucleotides comprising backbone modifications,branchpoints and non-nucleotide residues, groups or bridges. Identifyingsuitable nucleic acid aptamers basically involves selecting aptamersthat bind specifically to one or more of the above-identified biomarkersthat contain the nuclei-associated epitopes with sufficiently highaffinity (e.g., Kd=20-50 nM) and specificity from a pool of nucleicacids containing a random region of varying or predetermined length (Shiet al., “A Specific RNA Hairpin Loop Structure Binds the RNA RecognitionMotifs of the Drosophila SR Protein B52,” Mol. Cell Biol. 17:1649-1657(1997); Shi, “Perturbing Protein Function with RNA Aptamers” (thesis,Cornell University) microformed on (University Microfilms, Inc. 1997),each of which is hereby incorporated by reference in their entirety).This procedure is known as SELEX. The SELEX scheme is described indetail in U.S. Pat. No. 5,270,163 to Gold et al.; Ellington and Szostak,“In Vitro Selection of RNA Molecules that Bind Specific Ligands,” Nature346:818-822 (1990); and Tuerk & Gold, “Systematic Evolution of Ligandsby Exponential Enrichment: RNA Ligands to Bacteriophage T4 DNAPolymerase,” Science 249:505-510 (1990), each of which is herebyincorporated by reference in its entirety.

Nucleic acid aptamers include multivalent aptamers and bivalentaptamers. Methods of making bivalent and multivalent aptamers aredescribed in U.S. Pat. No. 6,458,559 to Shi et al., which is herebyincorporated by reference in its entirety. A method for modular designand construction of multivalent nucleic acid aptamers, their expression,and methods of use are described in U.S. Patent Publication No.2005/0282190 to Shi et al, which is hereby incorporated by reference inits entirety.

NAESA/L are preferably labeled with a dye or fluorophore that exhibits adistinct spectral emission relative to the NAD, allowing the NAES/Llabel to be discriminated from the NAD emissions. Exemplary dyes orfluorophores include, without limitation, ALEXA® dyes from LifeTechnologies, DYLIGHT™dyes from Norus Biologics, CF™ dyes available fromBiotium Inc., and fluorochromes like FITC and PE. The NAESA/L can beintroduced in any amount that is effective to label the epitope ofinterest, typically about 0.5 μg/ml to about 5.0 μg/ml.

In one embodiment of the methods of the present invention, contactingthe sample with one or more lysis solutions and contacting the freenuclei and/or chromatin debris with RNase (optional), a NAD, and one ormore NAESA/Ls is carried out simultaneously. Alternatively, these stepsare carried out sequentially.

Suitable NAD and NAESA/L reagents are capable of labeling cellular DNAand/or nuclei-associated epitopes of interest at a concentration rangedetectable by flow cytometry, image analysis, and/or laser-scanningcytometry, and have fluorescent emission spectrums that do notsubstantially overlap. It should be appreciated by those of ordinaryskill in the art that additional fluorescent NAD and NAESA/L reagentsare known and are continually being identified. Any suitable reagentwith requisite binding specificity and fluorescence characteristics canbe employed.

Single-laser flow cytometric analysis uses a single focused laser beamwith an appropriate emission band to excite the NAD and NAESA/Lfluorochromes. As labeled nuclei, chromatin debris, and optionalcounting beads pass through the focused laser beam, they exhibit afluorescent emission maxima characteristic of the fluorescent reagent(s)associated therewith. Dual- or multiple-laser flow cytometric analysisuses two or more focused laser beams with appropriate emission bands inmuch the same manner as described for the single-laser flow cytometer.Different emission bands afforded by the two or more lasers allow foradditional combinations of fluorescent reagents to be employed, andrepresents a preferred analytical platform for conducting the assay whenseveral fluorochromes are utilized.

Preferably, the flow cytometer is equipped with appropriate detectiondevices to enable detection of the fluorescent emissions and lightscatter produced by the nuclei and chromatin debris and, if used,counting beads. These “light scatter” signals serve as additionalcriteria that help discriminate nuclei, chromatin debris, and countingbeads from one another.

A further aspect of the present invention relates to a method ofassessing a singular nuclear biomarker for its responsiveness toexternal stimuli, for instance a chemical or physical agent. This methodinvolves exposing eukaryotic cells to a chemical or physical agent. In apreferred embodiment of the methods of the present invention, the cellsare then contacted with a lysis solution that includes RNase, a NAD, andone NAESA/L reagent. Samples are then analyzed for the number of nuclei,the number of NAESA/L-positive nuclei, mean and/or medianNAESA/L-associated fluorescence intensity, and optionally the number ofchromatin debris, the number of NAESA/L-positive chromatin debris, thenumber of nuclei in G1, S, and G2/M phases of the cell cycle, the numberof polyploid nuclei, and the number of counting beads. A significantdeviation in these values, especially the frequency of NAESA/L-positiveevents and/or mean NAESA/L-associated fluorescence intensity relative toa baseline value in unexposed or negative control eukaryotic cells,indicates the chemical or physical agent's ability to affect the amountand/or activity of the nuclear biomarker being studied.

Physical agents that are known to affect DNA and/or othernuclei-associated epitopes include, without limitation, heat, cold,gamma, alpha, and beta radiation, and UV radiation.

Chemical agents which are known to affect DNA and/or othernuclei-associated epitopes include, without limitation, tumor promotersincluding phorbol esters and dioxin-like chemicals, inorganicgenotoxicants (e.g., arsenic, cadmium and nickel), organic genotoxicants(for instance those used as antineoplastic drugs, such ascyclophosphamide, cisplatin, vinblastine, cytosine arabinoside, andothers), anti-metabolites (for instance those used as antineoplasticdrugs, such as methotrexate and 5-fluorouracil), organic genotoxicantsthat are generated by combustion processes (for instance polycyclicaromatic hydrocarbons such as benzo(a)pyrene), certain protein kinaseinhibitors, as well as organic genotoxicants that are found in nature(e.g., aflatoxins such as aflatoxin B1).

A further aspect of the present invention relates to a method ofassessing multiple nuclear biomarkers for their responsiveness toexternal stimuli, for instance a chemical or physical agent. This methodinvolves exposing eukaryotic cells to a chemical or physical agent. In apreferred embodiment of the methods of the present invention, the cellsare then contacted with a lysis solution that includes RNase, a NAD, andone or more NAESA/L reagents. Based on the number of nuclear biomarkersbeing investigated and the capabilities of the analytical detectionequipment, it may be necessary to consider each of the multiple NAESA/Lsone at a time (i.e., over several parallel samples) in order tooptimally detect each NAESA/L. Alternatively, the multiple NAESA/Ls canbe detected simultaneously following their use in a single sample.Samples are analyzed for the number of nuclei, the number ofNAESA/L-positive nuclei, mean and/or median NAESA/L-associatedfluorescence intensity, and optionally the number of chromatin debris,the number of NAESA/L-positive chromatin debris, the number of nuclei inG1, S, and G2/M phases of the cell cycle, the number of polyploidnuclei, and the number of counting beads. A significant deviation inthese values, especially the frequencies of NAESA/L-positive eventsand/or mean NAESA/L-associated fluorescence intensity relative tobaseline values in unexposed or negative control eukaryotic cells,indicates the chemical or physical agent's ability to affect the amountand/or activity of the nuclear biomarkers being studied.

A further aspect of the present invention relates to a method ofassessing multiple nuclear biomarkers for their responsiveness toexternal stimuli, for instance a chemical or physical agent, and usingthe resulting information to classify the agents according to predefinedcategories. This method involves exposing eukaryotic cells to a chemicalor physical agent. In a preferred embodiment of the methods of thepresent invention, the sample is contacted with a lysis solution thatincludes RNase, a NAD, and one or more NAESA/L reagents. Based on thenumber of nuclear biomarkers and the capabilities of the analyticaldetection equipment, it may be necessary to consider each of themultiple NAESA/Ls one at a time (i.e., over several parallel samples) inorder to optimally detect each NAESA/L. Alternatively, the multipleNAESA/Ls can be detected simultaneously following their use in a singlesample. Samples are analyzed for the number of nuclei, the number ofNAESA/L-positive nuclei, mean and/or median NAESA/L-associatedfluorescence intensity, and optionally the number of chromatin debris,the number of NAESA/L-positive chromatin debris, the number of nuclei inG1, S, and G2/M phases of the cell cycle, the number of polyploidnuclei, and the number of counting beads. A significant deviation inthese values, especially the frequencies of NAESA/L-positive eventsand/or mean NAESA/L-associated fluorescence intensity relative tobaseline values in unexposed or negative control eukaryotic cells,indicates the chemical or physical agent's ability to affect the amountand/or activity of the nuclear biomarkers being studied. By consideringthe biomarkers' responsiveness and/or response magnitude, a statisticalmethod is used to predict a category that best describes the chemical orphysical agent. In a preferred embodiment of the present invention,logistic regression and/or discriminant analysis is used to predict acategory and provide a measure of confidence associated with theprediction.

A further aspect of the present invention relates to a method ofassessing multiple nuclear biomarkers for their responsiveness toexternal stimuli, for instance a chemical or physical agent, and usingthe resulting information to classify the agents according to genotoxicmode of action. This method involves exposing eukaryotic cells to achemical or physical agent. In a preferred embodiment of the methods ofthe present invention, the sample is contacted with a lysis solutionthat includes RNase, a NAD, and one or more NAESA/L reagents. Based onthe number of nuclear biomarkers and the capabilities of the analyticaldetection equipment, it may be necessary to consider each of themultiple NAESA/Ls one at a time (i.e., over several parallel samples) inorder to optimally detect each NAESA/L. Alternatively, the multipleNAESA/Ls can be detected simultaneously following their use in a singlesample. Samples are analyzed for the number of nuclei, the number ofNAESA/L-positive nuclei, mean and/or median NAESA/L-associatedfluorescence intensity, and optionally the number of chromatin debris,the number of NAESA/L-positive chromatin debris, the number of nuclei inG1, S, and G2/M phases of the cell cycle, the number of polyploidnuclei, and the number of counting beads. A significant deviation inthese values in unexposed or negative control eukaryotic cells isindicative of the chemical or physical agent's genotoxic mode of action.By considering the biomarkers' responsiveness and/or response magnitude,a statistical method is used to predict a category to describe thechemical or physical agent. In a preferred embodiment of the presentinvention, logistic regression and/or discriminant analysis is used topredict a category, for example cytotoxic, genotoxic with a clastogenicMOA, or genotoxic with an aneugenic MOA, and provide a measure ofconfidence associated with the prediction.

In the several aspects of the invention, particularly preferredcombinations of reagents have been identified that are capable of highlyselective discrimination of cytotoxicity, genotoxicity with clastogenicmode of action, and genotoxicity with aneugenic mode of action.Preferably, each of these combinations includes, without limitation, aNAD, a first NAESA/L that binds specifically a nuclei-associated epitopeassociated with double-strand DNA breaks; a second NAESA/L that isresponsive to aneugenic activity; and a third reagent that is either aNAESA/L that binds specifically to an epitope associated withcytotoxicity or a dye responsive to cytotoxicity. Particularly preferredcombinations of reagents include, without limitation, (i) a NAD, a firstNAESA/L that binds specifically to an epitope present on phosphorylatedγH2AX, a second NAESA/L that binds specifically to an epitope present onphosphorylated H3, and a third NAESA/L that binds specifically to anepitope present on cleaved caspase 3 or cleaved PARP; (ii) a NAD, afirst NAESA/L that binds specifically to an epitope present onphosphorylated γH2AX, a second NAESA/L that binds specifically to anepitope present on phosphorylated H3, and a DNA dye that penetrates deadand dying cells, but not viable cells, such as the photoactivatable dyesethidium monoazide bromide or propidium monoazide bromide; (iii) a NAD,a first NAESA/L that binds specifically to an epitope present onphosphorylated γH2AX, a second NAESA/L that binds specifically to anepitope present on phosphorylated H3, and a mitochondrial membranepotential dye such as TMRE; (iv) a NAD, a first NAESA/L that bindsspecifically to an epitope present on phosphorylated γH2AX, a secondNAESA/L that binds specifically to an epitope present on phosphorylatedH3, and a reagent that measures cellular ATP levels (e.g., a luminescentreagent such as luciferase/D-luciferin); (v) a NAD, a first NAESA/L thatbinds specifically to an epitope present on cleaved PARP, and a secondNAESA/L that binds specifically to an epitope present on cleaved caspase3 and/or 7 and/or 9; and (vi) a NAD, a first NAESA/L that bindsspecifically to an epitope present on phosphorylated-H3, and either asecond NAESA/L that binds specifically to an epitope present on Ki-67 orPCNA, or the dye CFSE, which are markers of cell proliferation. For eachof these combinations, in certain embodiments the NAD and NAESA/L areprovided in a single lysis solution that also includes RNase andcounting beads.

In these several methods for screening the effects of chemical orphysical agents on the single or multiple nuclear biomarkers, thesemethods can be carried out in parallel with, or subsequent to, ananalysis according to the methods described in U.S. Pat. Nos. 7,445,910and 7,645,593 to Dertinger et al., which are hereby incorporated byreference in its entirety, or the methods described in U.S. PatentApplication Publ. No. 20140017673 to Dertinger et al., which is herebyincorporated by reference in its entirety. For instance, the methods forscoring an in vitro micronucleus assay as described in these patentreferences can be used to characterize treatment-related cytotoxicity orgenotoxicity, and in the case of the above-identified patent applicationcharacterize whether the genotoxic activity is the result of ananeugenic or clastogenic mode of action.

In carrying out the methods of the present invention, exposure ofeukaryotic cells to physical or chemical agents is preferably carriedout for a predetermined period of exposure time. Preferred exposuretimes will depend on the target being studied. For example, whenconsidering chemicals for their genotoxic potential, exposure timestypically range from about 3 hrs to the equivalent of approximately twopopulation doublings.

Methods of assessing the responsiveness of nuclear biomarkers tophysical or chemical agents may further involve a delay between the endof exposure and prior to performing cell harvest, membrane lysis,labeling, and analysis according to the previously described methods ofthe present invention. When employed, the delay or “recovery” period ispreferably between about 1 minute and the equivalent of approximatelytwo population doublings, although longer or shorter delays can also beutilized.

To some degree, exposure time and recovery periods will be cell line-and nuclear biomarker-dependent. Persons of skill in the art can readilyoptimize the methods of the present invention for different types ofeukaryotic cells and different physical or chemical agents.

Certain agents may offer protection from adverse stimuli, while othersmagnify deleterious effects. The present invention can be used toevaluate the effects of an agent that can modify (i.e., enhance orsuppress) such responses. To assess the suspected protective effects ofan agent, it can be added to the culture of cells prior to, concurrentlywith, or soon after addition of a known stressor. Any protective effectafforded by the agent can be measured relative to damage caused by thestressor agent alone. For example, putative protective agents can bevitamins, bioflavonoids and anti-oxidants, dietary supplements (e.g.,herbal supplements), or any other protective agent, naturally occurringor synthesized by man.

To assess the ability of an agent to synergistically or additivelyenhance adverse effects, the agent can be added to the culture of cellsprior to, concurrently with, or shortly after addition of a knownstressor. Any additive or synergistic effect caused by the agent can bemeasured relative to deleterious effects caused by either stressor agentalone.

Yet another aspect of the present invention relates to a kit thatincludes: one or more eukaryotic cell membrane lysis solutions; RNase, aNAD, and instructions for their use. Kits are also provided that may ormay not lack one or more of the preceding components (e.g., RNase isoptional), and may include one or more of the following components:NAESA/L that recognize specific nuclei-associated epitope(s),instructions for the use of these reagents, and/or a computer-readablemedium that contains instructions for statistical analyses forcharacterizing in vitro genotoxicity as the result of clastogenicity,aneugenicity, or cytotoxicity based on the behavior of a eukaryotic cellin response to exposure to a chemical or physical agent and followingexposure to the kit components. These statistical analyses may includelogistic regression and/or discriminant analysis. Ideally, thefluorescent emission spectra of the NAD and NAESA/L do not substantiallyoverlap.

In certain embodiments, the one or more eukaryotic cell membrane lysissolution(s) also contain(s) all other reagents supplied with the kit, inwhich case the kit contains the one or more solutions, preferably asingle solution, and optionally the computer-readable medium, andinstructions for their use. Thus, a further aspect of the presentinvention relates to an analytical formulation in the form of an aqueoussolution that includes: (i) an effective amount of one or more agentsfor causing eukaryotic cell membrane lysis, preferably about 0.1% toabout 10% of a non-ionic surfactant of the type described above; (ii) aneffective amount of an RNase, preferably about 0.05 mg/ml to about 5.0mg/ml; and (iii) an effective amount of a NAD, preferably about 0.1μg/m1 to about 15 μg/ml. In an alternative embodiment, the analyticalformulation may also include (iv) counting beads, preferably in anamount affording a final concentration of about 500 beads/ml to about50,000 beads/ml. In an alternative embodiment, the analyticalformulation may also include, with or without the counting beads, (v) aneffective amount of one or more, preferably two or more, or even threeor more NAESA/L against distinct nuclei-associated epitopes and/or (vi)an effective amount of a fluorescent or luminescent reagent that isresponsive to cytotoxicity, as described above. Effective amounts foreach of the one or more NAESA/L is preferably about 0.5 μg/m1 to about 5μl/ml. In yet another embodiment, the analytical formulation includescomponents (i)-(v) as identified above, or (i)-(vi) as identified above.Finally, for each of the preceding embodiments identified above, wherethe NAD is a DNA-specific dye, then the effective amount of RNase,component (ii), can be omitted from each of the analytical formulations.In alternative embodiments, the analytical formulation consists ofcomponents (i), (iii), (iv); components (i), (iii)-(v); or components(i), (iii)-(vi); any of which may optionally contain component (ii), oneor more buffers, one or more stabilizers, and/or one or morepreservatives added to the composition.

EXAMPLES

The examples below are intended to exemplify the practice of the presentinvention but are by no means intended to limit the scope thereof.

Cells and Culture Medium

The TK6 cells used in these experiments were from American Type TissueCollection (ATCC) (Manassas, Va.). Cells were maintained in culturemedium at 37° C., 5% CO₂, and in a humid atmosphere. Cells weremaintained between approximately 1×10⁴ and 1×10⁶ cells/ml for routinepassage. The culture medium consisted of RPMI 1640 supplemented with 2mM L-glutamine, 100 IU penicillin and 100 μg/m1 streptomycin, to whichheat inactivated horse serum was added for 10% v/v final concentration(all from MediaTech Inc., Herndon, Va.).

Treatment of Cells with Toxic Agents

For these experiments, TK6 cells were treated with solvent (most oftenDMSO, 1% v/v) and a range of closely spaced test article concentrations.Treatments occurred in wells of a 96-well plate. At the start oftreatment, cells were at 2×10⁵/ml in a volume of 300 μl per well.Counting beads (PEAK FLOW™) were included in these cultures at aconcentration of approximately 1 drop per 10 ml, and served as a meansto derive relative nuclei to bead ratios (relative to solvent controls).Continuous treatment occurred for approximately 24 hrs, during whichtime plates were incubated at 37° C., 5% CO₂, and in a humid atmosphere.Each test article concentration was studied in 3 to 6 replicate wells.

The γH2AX endpoint described herein was studied 4 and/or 24-27 hrs afterstart of treatment. Each of the remaining endpoints were studied at theconclusion of the treatment period, that is 24 to 27 hrs afterinitiation of exposure to test article (which for this cell linecorresponds to approximately 1.5 to 2.0 population doubling times).

Liberating and Labeling Nuclei for Flow Cytometric Analysis

At the time of cell harvest (4 hrs and 24 to 27 hrs), cells wereresuspended with gentle pipetting and 25 μl aliquots were transferred towells of a 96 well plate containing 50 μl pre-aliquoted lysis solution.Lysis solution was composed of deionized water, 0.584 mg NaCl/ml, 1 mgsodium citrate/ml, 0.6 μl IGEPAL®/ml, 1 mg RNase A/ml, NAD (0.4 μMSYTOX® Green), counting beads, and as described in more detail below,one of the following NAESA/L reagents: anti-cleaved caspase 3-ALEXA® 647antibody (5 μl/ml) (Cleaved Caspase-3 (Asp175) (D3E9) Rabbit mAb (AlexaFluor® 647 Conjugate), Cell Signaling Technology, Danvers, Mass.) oranti-cleaved PARP-ALEXA® 647 (5 μl/ml) (Alexa Fluor® 647 Mouseanti-Cleaved PARP (Asp 214), BD Biosciences, San Jose, Calif.) antibodyor anti-γH2AX-ALEXA® 647 antibody (5 μl/ml) (Alexa Fluor® 647 Mouseanti-H₂AX (pS139), BD Biosciences, San Jose, Calif.) or anti-H3-ALEXA®647 antibody (5 μl/ml) (Alexa Fluor® 647 Rat anti-H3 (pS28), BDBiosciences, San Jose, Calif.).

After incubation at room temperate for at least 5 minutes, samples wereanalyzed with a dual-laser flow cytometer, 488 nm and 633 nm excitation(FACSCanto II, BD Biosciences, San Jose, Calif.). Instrumentationsettings and data acquisition/analysis were controlled with Divasoftware v6.1.3. SYTOX® Green-associated fluorescence emission wascollected in the FITC channel, and ALEXA® 647-associated fluorescencewas collected in the APC channel. The BD Biosciences High ThroughputSampler (HTS) attachment was used to automatically analyze each of thesamples in the 96 well plate(s). For the majority of these endpoints andexperiments, the stop mode was configured such that each well wasanalyzed for 5 seconds, which usually represented sufficient time toacquire at least 500 NAD-positive nuclei.

Micronucleus Assay

The in vitro micronucleus assay is a chromosomal damage test that iswidely used to evaluate chemical and/or physical agents for clastogenicand aneugenic activity. While the sensitivity of the micronucleus testis generally regarded as high, there are ongoing concerns that this andother eukaryotic cell in vitro genotoxicity assays exhibit unacceptablylow specificity (i.e., high false positive rates). Furthermore, in theusual conduct of the in vitro micronucleus assay, the discrimination ofbona fide genotoxic activity as primarily occurring through aclastogenic or aneugenic mode of action is not readily obtained. Example4 describes experiments with 20 diverse chemical agents that exhibitedpositive in vitro micronucleus results. (Clastogens: arabinofuranosylcytidine, etoposide, methyl methanesulfonate, cisplatin, camptothecin,aphidicoline, 5-fluorouracil, 4-nitroquinoline-1-oxide, and hydroxyurea;aneugens: vinblastine, paclitaxol, carbendazim, griseofulvin, anddiethylstilbestrol; cytotoxicants: carbonyl cyanide m-chlorophenylhydrazone, anti-FAS, nutlin-3, tunicamycin, phenformin HC1, andtributyltin.) The methodology used to score micronuclei in cultures ofTK6 cells was commercially available In Vitro MICROFLOW® kits (LitronLaboratories, Rochester, N.Y.). Given continuous exposure to thesechemicals (24-27 hrs), 13 of the positive results were expected based ontheir known clastogenic or aneugenic activity. However, 4 positivefindings were regarded false or irrelevant positives, likelyattributable to DNA damage that was secondary to cytotoxicity as opposedto DNA-reactivity. These chemicals therefore represented a goodopportunity to explore whether one or more nuclear biomarkers, asstudied according to present invention, could be used to categorizethese in vitro genotoxic-positive compounds as clastogenic, aneugenic,or cytotoxic.

For these experiments, each of the 20 chemicals was tested over closelyspaced concentrations, 6 replicate wells per concentration. Three wellswere used for the micronucleus analyses, and three wells were used forthe several nuclear biomarker assessments of the type described inExample 1. Other endpoints of cytotoxicity were considered, includingNAD-positive nuclei to time ratios, ethidium monoazide bromide (EMA)positive events as an indicator of membrane integrity,tetramethylrhodamine ethyl ester (TMRE) negative cells as an assessmentof mitochondrial membrane potential, and ATP levels (using luciferase)as an assessment of overall cell health in the sample. We believed thatperhaps one but more likely two or more of these nuclei biomarker orother endpoints would provide signatures that would enable us toaccurately classify these chemicals into groups according to MOA.Further details are described under Statistical Analyses, as well asExample 4 below.

Statistical Analyses

Logistic regression is considered a part of generalized linear models.Logistic regression allows one to predict a discrete outcome, such asgroup membership, from a set of explanatory variables that may becontinuous, discrete, dichotomous, or any combination of these.Discriminant analysis is a related statistical method that can also beused to predict group membership. In some circumstances there areadvantages to logistic regression, because it makes fewer assumptionsabout the explanatory variables, for instance it does not require normaldistribution or equal variances. In Example 4 which follows, thelogistic regression platform (JMP software, v 8.0.1, SAS Institute,Inc.) was utilized to construct a model whereby nuclei biomarker and/orother cytotoxicity endpoints were considered for their ability topredict class of chemical agent, that is, either clastogenic, aneugenic,or cytotoxic. This categorization strategy has many potentialapplications, including following up a positive in vitro chromosomaldamage assay result to determine genotoxic MOA.

To build the model, individual explanatory variables, described in moredetail in Example 4, were tested for their ability to categorizechemicals into one of three groups. In an iterative process, significantexplanatory variables were added to a model with the goal of buildingthe most parsimonious model that correctly categorized each of the 20chemicals into groups specified a priori.

Example 1 Nuclear Biomarker γH2AX is Responsive to Clastogenic Activity

In this experiment, TK6 cells were treated with the prototypicalclastogenic agent methyl methanesulfonate (MMS), or the cytotoxicantanti-FAS. Treatments occurred over a range of closely spacedconcentrations. Approximately 24-27 hours after the start of treatment,25 μl aliquots of cells were added to lysis solution containing RNase,NAC, anti-γH2AX-ALEXA® 647, and counting beads. As shown by Table I,each chemical caused concentration-dependent reductions to nuclei tobead ratios, indicative of fewer cells and therefore cytotoxicity.Whereas the clastogen MMS was observed to cause higheranti-γH2AX-associated fluorescence intensity relative to solvent controlwells, no change was evident with the cytotoxicant anti-FAS. See FIG. 1for representative bivariate plots that illustrate the regions used tomake these measurements.

TABLE I Shift in γH2AX-associated Fluorescence in Response to aClastogen Nuclei to Test Article Bead Ratio γH2AX Fluorescence TestArticle Conc. (% of Control) (Fold-Increase)^(†) Solvent Control 0 1001.00 MMS 2.344 μg/ml 88 3.13 MMS 3.125 μg/ml 87 4.11 MMS 4.6875 μg/ml 834.36 MMS 6.25 μg/ml 78 4.92 MMS 9.375 μg/ml 64 4.79 MMS 12.5 μg/ml 584.80 Anti-FAS 9.375 ng/ml 87 0.93 Anti-FAS 12.5 ng/ml 86 0.92 Anti-FAS18.75 ng/ml 80 0.94 Anti-FAS 25 ng/ml 71 0.95 Anti-FAS 37.5 ng/ml 660.98 Anti-FAS 50 ng/ml 50 1.00 ^(†)Fold Increase is relative to Control

Example 2 Nuclear Biomarkers PARP and Cleaved Caspase 3 are Responsiveto Apoptotic Activity

In this experiment, TK6 cells were treated with the prototypicalapoptosis-inducing agent carbonyl cyanide m-chlorophenyl hydrazone(CCCP). Treatments occurred over a range of closely spacedconcentrations. Approximately 24-27 hours after the start of treatment,25 μl aliquots of cells were added to lysis solutions containing RNase,NAC, and either anti-cleaved PARP-ALEXA® 647 or anti-cleaved Caspase 3.As shown by Table II, CCCP caused concentration-dependent reductions tonuclei to bead ratios, indicative of fewer cells and thereforecytotoxicity. This chemical was also observed to affect cleaved PARP andCaspase 3 levels in nuclei, as evidenced by the higher values of cleavedPARP- and Caspase 3-positive nuclei relative to solvent control wells.See FIG. 2 for representative bivariate plots that illustrate theregions used to make these measurements.

TABLE II Cleaved PARP- and Caspase 3-Positive Nuclei in Response to theCytotoxicant CCCP Nuclei to Bead PARP-Positive Caspase 3-Positive CCCPConc. Ratio Nuclei Nuclei (μM) (% of Control) (Fold-Increase)^(†)(Fold-Increase)^(†) 0 100 1.00 1.00 3.125 79 4.90 2.63 4.6875 69 8.604.33 6.25 64 12.1 5.86 9.375 59 20.75 9.13 12.5 50 34.45 18.83 ^(†)FoldIncrease is relative to Control

Example 3 Nuclear Biomarker phospho-H3 is Responsive to AneugenicActivity

In this experiment, TK6 cells were treated with the prototypical aneugenvinblastine and the prototypical clastogen methyl methanesulfonate(MMS). Treatments occurred over a range of closely spacedconcentrations. Approximately 24-27 hours after the start of treatment,25 μl aliquots of cells were added to lysis solutions containing RNase,NAC, anti-H3-ALEXA® 647 (recognizes phospho-serine 28), and countingbeads. As shown by Table III, vinblastine and MMS causedconcentration-dependent reductions to nuclei to bead ratios, indicativeof fewer cells and therefore cytotoxicity. Whereas the aneugenvinblastine was observed to markedly increase the frequencies ofH3-positive nuclei, the clastogen had no such effect. See FIG. 3 forrepresentative bivariate plots that illustrate the regions used to makethese measurements.

TABLE III Phospho-H3-Positive Nuclei in Response to Vinblastine and MMSNuclei to Bead Phospho-H3+ Ratio Nuclei Test Article Test Article Conc.(% of Control) (Fold-Increase)^(†) Solvent Control 0 100 1 Vinblastine0.3 ng/ml 98 .86 Vinblastine 0.4 ng/ml 97 .86 Vinblastine 0.6 ng/ml 861.23 Vinblastine 0.8 ng/ml 81 2.11 Vinblastine 1.2 ng/ml 71 4.85Vinblastine 1.6 ng/ml 58 5.41 MMS 2.344 μg/ml 88 1.00 MMS 3.125 μg/ml 870.76 MMS 4.6875 μg/ml 83 0.67 MMS 6.25 μg/ml 78 0.71 MMS 9.375 μg/ml 640.76 MMS 12.5 μg/ml 58 0.65 ^(†)Fold Increase is relative to Control

Example 4 Matrix of Biomarkers Predict Genotoxic Mode of Action

In these experiments, TK6 cells were treated with each of 9 clastogens,5 aneugens, and 6 cytotoxicants. Each of these agents was observed inprevious experiments to induce what appeared to be significantmicronucleus responses at concentrations that were deemed moderately butnot overly cytotoxic. Those cytotoxicity evaluations were based on aconventional approach for assessing cytotoxicity in genotoxicity tests,that is, relative cell counts (which in the case of the flowcytometry-based In Vitro MICROFLOW® kit is accomplished by measuringnuclei to counting bead ratio or else nuclei to time ratio andexpressing these values relative to the mean solvent control value). Forthe follow-up experiments, treatments occurred over a range of closelyspaced concentrations. Approximately 24-27 hours after the start oftreatment, three wells per concentration were processed via MICROFLOW®instructions for micronucleus frequencies, relative nuclei to timevalues, and EMA-positive events (an assessment of membrane integrity).The remaining three wells per concentration were used to accomplish aseries of nuclei biomarker readings according to the invention, as wellas two other assessments of cytotoxicity—frequency of TMRE-negativecells and ATP levels (via Promega's CELL TITERGLO® kit).

The resulting micronucleus data confirmed the initial experiments byshowing significant induction of micronuclei or what appeared to bemicronuclei at moderately cytotoxic treatment conditions as evaluated bynuclei to time ratios. (Induction of micronuclei was consideredsignificant when mean fold-increase relative to solvent control was≧3-fold; moderate but not excessive cytotoxicity was indicated byrelative nuclei to time ratios that approached but did fall below 45%.)These results confirm the high sensitivity but low specificity of the invitro micronucleus assay as currently practiced.

As indicated above, a series of nuclear biomarkers and other endpointswere evaluated for three parallel wells per concentration. In order toreduce the data-rich dose-response data for each experimental endpointto a single value, two approaches were tested. Each endpoint's meanvalue corresponding to the lowest effective micronucleus-inducingconcentration was recorded. Additionally, each endpoint's mean valuecorresponding to the highest passing concentration (i.e., highestconcentration that approached but did not fall below 45% relative nucleito time ratio) was recorded. These values, together with our a prioriclassification for each chemical noted above (i.e., clastogen, aneugen,or cytotoxicant) were analyzed using JMP's logistic regression platform.One initial observation was that more explanatory variables were foundto be statistically significant (p<0.05) when the data reduction processoccurred according to highest passing concentration. Furthermore, whenexplanatory variables were significant for both lowestmicronucleus-inducing effect concentration as well as highest passingconcentration, the latter tended to exhibit lower p values and higher R²values (indicative of a model's goodness of fit). Therefore, thesubsequent analyses that are described in more detail below weregenerated with the data reduction scheme that was based on the highestpassing concentration.

Table IV shows p and R² values, as well as the proportion of correctpredictions made by each of the explanatory variables in isolation.Whereas some endpoints were found to be quite effective at classifyingone or more of the categories, none was perfectly effective forclassifying across all three. The combination of γH2AX fluorescenceshift and fold-increase in phospho-H3-positive events was considered aparticularly complementary pair of endpoints, as the former isconsidered an indicator of double-strand DNA breaks and indeed was shownto be effective at classifying clastogens, while the latter is known tobe responsive to aneugenicity and showed good potential for detectingagents with this activity. As evidenced by a higher R² value, thecombination of these two nuclear biomarkers was observed to markedlyimprove the model. However, it was only when these two explanatoryvariables were combined with a third endpoint that can be generalized asbeing responsive to cytotoxicity that the most effective model wasrealized.

TABLE IV Logistic Regression Output for Classifying In VitroMicronucleus- Positive Events According to Mode of Action ClastogensAneugens Cytotoxicants Explanatory Correctly Correctly CorrectlyVariable(s) p value R² Predicted Predicted Predicted Notes γH2AX shift(4 hr) <0.0001 0.4621 8/9 5/5 6/6 Fairly effective across groups γH2AXshift 0.0085 0.2236 7/9 0/5 0/6 Lower predictivity (24-27 hr) relativeto 4 hr time point Phospho-H3+ <0.0001 0.5497 7/9 5/5 3/6 Tends tomisclassify cytotoxicants as clastogens Cleaved Cas3+ 0.0061 0.2387 7/90/5 4/6 Poor ability to classify aneugens Cleaved PARP+ 0.0014 0.30858/9 1/5 3/6 Poor ability to classify aneugens and cytotoxicants TMRE−cells <0.0001 0.4761 9/9 2/5 4/6 Poor ability to classify aneugens EMA+events 0.0006 0.3455 9/9 4/5 4/6 Fairly effective across groups ATPlevel <0.0001 0.6368 8/9 3/5 5/6 Fairly effective across groups γH2AXshift (4 hr) <0.0001 0.8325 8/9 5/5 6/6 One clastogen Phospho-H3+misclassified as cytotoxicant γH2AX shift (4 hr) <0.0001 1.0000 9//9 5/56/6 Zero misclassified Phospho-H3+ Cleaved Cas3+ γH2AX shift (4 hr)<0.0001 1.0000 9//9 5/5 6/6 Zero misclassified Phospho-H3+ Cleaved PARP+γH2AX shift (4 hr) <0.0001 1.0000 9//9 5/5 6/6 Zero misclassifiedPhospho-H3+ TMRE− Cells γH2AX shift (4 hr) <0.0001 1.0000 9//9 5/5 6/6Zero misclassified Phospho-H3+ EMA+ Events γH2AX shift (4 hr) <0.00011.0000 9//9 5/5 6/6 Zero misclassified Phospho-H3+ ATP Level p value =the observed significance probability for the Chi-square test; R² = theproportion of the total uncertainty that is attributed to the model fit,“goodness of fit”

From the data above, it is reasonable to conclude that a matrix ofendpoints that include nuclei biomarkers measured according to methodsdescribed by this invention are able to distinguish between clastogens,aneugens, and cytotoxicants. Importantly, logistic regression andrelated tests are able to make these predictions for new chemicals byadding their data to the statistical program, but in those cases withoutspecifying a priori categories. The resulting models represent powerfultools, as their categorizations are each accompanied by measures ofprobability, that is, their confidence in a given call.

It appears as though the most effective and parsimonious model foraccurately categorizing in vitro micronucleus-positive chemicals intoone of three groups can be summarized as follows: i) include at leastone sensitive and specific endpoint of double-strand DNA breaks, forinstance γH2AX-associated fluorescence shift; and ii) include at leastone sensitive and specific endpoint that is responsive to aneugenicactivity, for instance frequency of phospho-H3-positive events; and iii)include at least one endpoint that is responsive to cytotoxicity, forinstance any one of the following—cleaved caspase 3-positive events,cleaved PARP-positive events, TMRE-negative cells, EMA-positive events,or ATP levels. While these and a multitude of other explanatoryvariables may be incorporated into such a model, it is known that overlycomplex models can fail for a variety of reasons, and the goal shouldtherefore be to reduce the model to its simplest yet effective form.

Example 5 Multiplex Assay

This experiment highlights the ability of the invention to combinemultiple nuclei-associated endpoints into one flow cytometric analysis.For this experiment, log-phase TK6 cells were treated with cisplatinover a wide range of concentrations and then incubated at 37° C. for 24hours. Each concentration was studied in triplicate wells of a 96 wellplate. After 24 hours, well contents were pipetted up and down severaltimes to form homogenous suspensions, and 25 μl per well was added towells of a new 96 well plate that contained 50 μl of workinglysis/labeling/staining solution. This lysis/labeling/staining solutionwas comprised of 5 μl anti-γH2AX-Alexa® 488/ml, 1 μlanti-phospho-H3-PE/ml, 5 μl anti-cleaved PARP-Alexa® 647/ml, 5 μlRNase/ml, 25 μl propidium iodide/ml, and 1202 CountBright™ AbsoluteCount Bead/50 ul

As shown in Table V below, the information collected in this analysisincludes but is not limited to: absolute and relative nuclei counts,which were observed to become reduced in a cisplatinconcentration-dependent manner; anti-cleaved PARP positive chromatin,which increased in a cisplatin concentration-dependent manner; shift inanti-γH2AX-associated fluorescence, which increased in a cisplatinconcentration-dependent manner; anti-phospho-H3 positive nuclei whichwas reduced in a cisplatin concentration-dependent manner, and otherchanges to cell cycle, which can be largely described as an accumulationof cells in the G2/M phase of the cell cycle.

TABLE V Multiplexed Analysis of Nuclei Count, H2AX, H3 and PARP inResponse to Cisplatin % Relative γH2AX- Phospho-H3+ Cleaved Proportionof Cisplatin Nuclei associated Shift Nuclei PARP + Nuclei Cells in G2M(μg/ml) Count (Fold Increase)^(†) (Fold Increase)^(†) (FoldIncrease)^(†) (Fold Increase)^(†) 0 100 1.00 1.00 1.00 1.0 0.0024 1111.01 0.91 0.92 1.3 0.0049 109 1.11 1.00 0.96 1.2 0.0098 105 1.16 0.900.99 1.2 0.0195 102 1.23 0.90 0.99 1.3 0.0391 100 1.30 0.98 1.00 1.30.0781 95 1.40 0.94 1.09 1.4 0.1563 91 1.52 0.80 1.26 1.7 0.3125 77 1.730.82 1.74 1.9 0.625 66 2.16 0.45 3.20 2.2 1.25 53 2.90 0.28 8.60 2.3 2.537 3.90 0.22 22.78 2.1 ^(†)Fold Increase is relative to Control

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

What is claimed is:
 1. A method for analyzing detergent-liberated nucleiand/or chromatin debris for nuclei-associated epitopes, the methodcomprising: contacting a sample containing eukaryotic cells with asolution comprising one or more cell lysis reagents, a fluorescentnucleic acid dye (NAD), and one or more fluorescent nuclei-associatedepitope-specific antibodies or ligands (NAESA/L) that specifically bindto one or more nuclei-associated epitopes of interest, the NAD and oneor more NAESA/L having distinct fluorescent emission spectra, saidcontacting being effective to digest eukaryotic cell cytoplasmicmembranes but not nuclear membranes, aggregate metaphase chromosomes ofa single cell into a single bundle of chromosomes, label chromatin withthe NAD, and label the one or more nuclei-associated epitopes with theone or more NAESA/L; exciting the NAD and one or more NAESA/L with lightof an appropriate excitation wavelength; detecting fluorescent emissionand light scatter produced by the nuclei and/or chromatin debris andcounting one or more of the following events: the number of nuclei, thenumber of nuclei positively labeled by the one or more NAESA/L, thenumber of chromatin debris, the number of chromatin debris positivelylabeled by the one or more NAESA/L, the number of nuclei in G1, S, andG2/M phases of the cell cycle, and the number of polyploid nuclei; anddetermining one or more of the following measurements: the frequency ofnuclei positively labeled by the one or more NAESA/L relative to totalnuclei, the frequency of chromatin debris positively labeled by the oneor more NAESA/L relative to total chromatin debris and/or total nuclei,the proportion of nuclei in G1, S, and G2/M phases of the cell cycle,the proportion of polyploid nuclei, and mean and/or median fluorescenceby the one or more NAESA/L.
 2. A method of assessing the toxicity of achemical or physical agent, said method comprising: exposing eukaryoticcells to a chemical or physical agent and performing the methodaccording to claim 1, wherein, in comparison to unexposed or negativecontrol eukaryotic cells, a significant change to the proportion of theone or more NAESA/L-positive nuclei relative to total nuclei and/orchange to the proportion of the one or more NAESA/L-positive chromatindebris relative to total chromatin debris and/or total nuclei indicatesthat the chemical or physical agent affected the nuclei-associatedepitope of interest; a significant change to the proportion of nuclei inone or more phases of the cell cycle and/or in the proportion ofpolyploid nuclei indicates that the chemical or physical agent induced agenotoxic mode of action; and a significant change in the one or moreNAESA/L-positive nuclei and/or chromatin debris indicate that thechemical or physical agent affected the nuclei-associated epitope ofinterest, whereby an effect of the chemical or physical agent on two ormore of these endpoints allows a determination of genotoxic mode ofaction.
 3. The method according to claim 2 wherein the two or moreendpoints comprise two or more of: an increase in the phosphorylatedγH2AX, which indicates clastogenic activity; an increase in thephosphorylated H3 or polyploidy nuclei, each of which indicatesaneugenic activity; an increase in cleaved PARP+ events, cleaved Cas 3+event, cleaved Cas 7+ event, cleaved Cas 9+ events, TMRE-events, orEMA/PMA+ events indicates cytotoxicity; and a reduction in nuclei tobead ratios or nuclei to time ratios, or ATP levels indicatescytotoxicity.
 4. The method according to claim 2 wherein thesignificance of a change in one or more of the endpoints is determinedby logistic regression or discriminant analysis to assign the chemicalor physical agent to one of two or more categories selected from thegroup of cytotoxic, genotoxic with a clastogenic mode of action, orgenotoxic with an aneugenic mode of action.
 5. A method for assessingthe response of nuclei-associated biomarkers of DNA damage and/ortranscription factor activation, activity, or expression levels and/orepigenetic modifications resulting from a chemical or physical agent,the method comprising: exposing eukaryotic cells, previously exposed toa chemical or physical agent, to a solution comprising one or more celllysis reagents, optionally RNase, a NAD, and one or more NAESA/L, theNAD and one or more NAESA/L having distinct fluorescent emissionspectra, said exposing being effective to digest eukaryotic cellcytoplasmic membranes but not nuclear membranes, aggregate metaphasechromosomes of a single cell into a single bundle of chromosomes, labelchromatin with the NAD, and label the one or more nuclei-associatedepitopes with the one or more NAESA/L; exciting the NAD and one or moreNAESA/L with light of an appropriate excitation wavelength; anddetecting fluorescent emission and light scatter produced by the nucleiand/or chromatin debris and counting one or more of the followingevents: the number of nuclei, the number of NAESA/L-positive nuclei, thenumber of chromatin debris, the number of NAESA/L-positive chromatindebris, the number of nuclei in G1, S, and G2/M phases of the cellcycle, and the number of polyploid nuclei; wherein changes to theproportion of NAESA/L-positive nuclei relative to total nuclei and/orchanges to the proportion of NAESA/L-positive chromatin debris relativeto total chromatin debris and/or total nuclei indicates that thechemical or physical agent affected the nuclear biomarker; or a changein mean and/or median fluorescence of the one or more NAESA/L for anexposed sample, compared to unexposed or negative control eukaryoticcells, indicates that the chemical or physical agent affected thenuclear biomarker.
 6. A kit comprising: one or more eukaryotic cellmembrane lysis solutions; a fluorescent nucleic acid dye; and one ormore fluorescent reagents that bind specifically to a nuclei-associatedepitope.
 7. The kit according to claim 6 further comprising one or moreof RNase; counting beads; a computer-readable medium comprisinginstructions for statistical analyses for characterizing in vitrogenotoxicity as the result of clastogenicity, aneugenicity, orcytotoxicity based on the behavior of a eukaryotic cell in response toexposure to a chemical or physical agent and following exposure to thekit components.
 8. The kit according to claim 6 wherein the one or morenuclei-associated epitopes is selected from the group consisting ofphosphorylated γH2AX, phospho-H3, cleaved PARP, cleaved caspase 3, 7, or9, ChK1 and ChK2, ATM, ATR, H2B, AhR, BRCA1, BRCA2, RAD51, p53, 5-methylcytidine, and 8-OHdG.
 9. The kit according to claim 6 wherein the one ormore nuclei-associated epitopes comprises (i) a first epitope associatedwith double-strand DNA breaks, (ii) a second epitope that is responsiveto aneugenic activity.
 10. The kit according to claim 9 wherein the oneor more nuclei-associated epitopes further comprises (iii) a thirdepitope associated with cytotoxicity.
 11. The kit according to claim 9further comprising a different fluorescent or luminescent reagent thatis responsive to cytotoxicity.
 12. The kit according to claim 10,wherein the first epitope is present on phosphorylated γH2AX, the secondepitope is present on phosphorylated histone3 (H3), and the thirdepitope is present on cleaved caspase 3 or cleaved PARP.
 13. The kitaccording to claim 11, wherein the first epitope is present onphosphorylated γH2AX, the second epitope is present on phosphorylatedH3, and the different fluorescent or luminescent reagent is selectedfrom the group of ethidium monoazide bromide, propidium monoazidebromide, TMRE, and luciferase/luciferin.